Study On Detoxification Metabolic Mechanism Of Swimming Crab Portunus Trituberculatus Exposed To Ammonia

Ammonia is a major threat factor in aquaculture ambient water in pond. In thisstudy, some key detoxification-related genes were cloned and characterized from thegills and hepatopancreas of swimming crab Portunus trituberculatus. The effects ofammonia on ammonia transportation indicators, key detoxification-related geneexpression and ammonia metabolic parameters were determined to detect theammonia detoxification metabolism mechanism in the swimming crab Portunustrituberculatus. The study mainly includes three bodies as follows:(1) Cloningcharacterization and expression analysis of key detoxification-related genes inswimming crab P. trituberculatus, including Rh protein and glutamate dehydrogenase(GDH);(2) Effects of ammonia on ammonia transportation pathway in swimmingcrab P. trituberculatus;(3) Effects of ammonia on ammonia metabolic pathway inswimming crab P. trituberculatus. The results are as follows:1Cloning, characterization and expression analysis of detoxification-relatedgenes in swimming crab P. trituberculatusThe partial cDNA sequences of Rh protein and GDH were cloned from the gillsand hepatopancreas in swimming crab P. trituberculatus using degenerate primer andRT-PCR methods. The partial cDNA of Rh protein was of612bp encoding204amino acids. The Rh protein amino acids showed86%-91%high similarity to othercrustacean Rh protein. In addition, the partial cDNA of GDH was of414bp encoding138amino acids. The GDH amino acids showed high similarity of92%-96%to othercrustacean GDH.2Effects of ammonia exposure on ammonia transportation pathway inswimming crab P. trituberculatusThe results indicate that significant effects of ammonia exposure were seen onrelated parameters of ammonia transportation in tissues of P. trituberculatus. Our results showed that ambient ammonia exposure had significant effects onhaemolymph ammonia content, levels of Rh protein, GDH and GS mRNAexpressionas well as levels of Gln and urea in tissues. No significant was found in normal groups(P<0.05). During1mg/L ammonia-N and5mg/L ammonia-N exposure, haemolymphammonia content was notably increased only at6h and well below ambient ammonialevels during the entire exposure course. In gills, GDH activity was higher than that incontrol group and increased gradually within the first12h then remained unchanged,while Rh protein and GDH mRNA expression levels as well as glutamine synthetase(GS) activity and mRNAexpression level showed a peak change and the highest leveloccurred at6h or12h, in which Rh protein and GDH mRNA expression levels werehigher than control but GS activity and mRNA expression level returned to controllevel at48h. Gln level increased significantly within the first6h and then remainedthe same and had significantly difference compared with the control. There was apositive relationship between glutamine content and ambient ammonia concentration.While in hepatopancreas, GDH activity and mRNA expression level were notableonly at6h or2h and no other significant was observed throughout the48h period ascompared with the control level. Rh protein mRNA expression level and GS activityand mRNA expression level showed peak changes in48h or24h and the highestlevel accrued at12h after then returned or significant higher than normal. Gln levelincreased significantly within the first2h then tended to be stable from6h andshowed positive relationship with ammonia concentration. In muscle, GDH activityincreased significantly within the first6h and then remained unchanged in5mg L-1ammonia-N group, in contrast, no significant changes were found in1mg L-1ammonia-N group. The mRNAexpression levels of Rh protein, GDH and GS showedpeak changes in48h and the highest level accrued at12h or24h after then returnedor significant higher than normal at48h. GS activity and Gln level increasedsignificantly in the first12h and24h respectively, then remained stable showing apositive correlation with ambient ammonia. In haemolymph, GDH activity increasedremarkably within the first6h then tended to be stable in5mg/L ammonia group and no other significant changes was found in1mg/L ammonia group. GS activity andGln levels increased significantly within the first12h, and then remained the sameshowing a positive correlation with ambient ammonia content. Therefore, wesuggested that ammnia transportation was not inhibited in P. trituberculatus exposedto high ambient ammonia. The crabs could convert ammonia to glutamine to detoxifyammonia in muscle during high ambient ammonia-N stress. Gln levels in tissuescould be used as important parameters to evaluate ammonia detoxification of P.trituberculatus under ambient ammonia-N stress.3Effects of ammonia exposure on ammonia metabolic pathway in swimmingcrab P. trituberculatusThe results indicate that significant effects of ammonia exposure were seen onrelated parameters of ammonia metabolic in tissues of P. trituberculatus. Our resultsshowed that ambient ammonia exposure had significant effects on tissues XDHmRNA expression level, ARG mRNA expression level and activity and contents ofuric acid and urea. No significant was found in normal groups (P<0.05).Uric acid wasonly detected in heamolymph and no xanthine oxidoreductase (XOR) activity wasmeasured. During1mg/L ammonia-N and5mg/L ammonia-N exposure, XDH andARG mRNA expression levels and ARG activity in gills showed a peak change in48h and the lowest or highest level occurred at2h or6h and2h, respectively, whichshowed lower or returned to control group. Urea contents increased significantly atthe whole sampling time and the highest values was observed at6h then bothremained unchanged after12h. While in hepatopancreas, the level mRNA expressionof XDH and ARG showed peak changes within exposure time and returned or higherthan control level at48h and the highest peak was found at12h or6h. ARG activityincreased notably at6h then remained unchanged, while urea content significantlyincreased at2h and then became stable. In muscle, the mRNA expression levels ofXDH and ARG, the activity of ARG and urea content also showed peak changes in48h or24h and the highest level was found at12h or6h, in which ARG activityincreased significantly within2h and became stable from12h showing a positive correlation with ambient ammonia content, while others returned or higher thannormal level at48h. The levels of uric acid, ARG activity and urea all showed peakchanges within24h and the highest levels were found at6h. After then, ARG activityreturned to control level while uric acid and urea content tended to be stable after24hammonia-N exposure and showed a positive correlation relationship with ambientammonia content. Therefore, the results suggested P. trituberculatus could convertammonia to uric acid and urea to lower ammonia accumulation during high ambientammonia-N stress. Uric acid synthesis was only inhibited in gills, while urea synthesiswas induced after ammonia exposure. In addition, uric acid and urea levels inheamolymph could be used as important parameters to evaluate ammoniadetoxification of P. trituberculatus under ambient ammonia-N stress.